Heterologous proteins expressed in transformed host cells occur either in a soluble form or in the form of inactive and insoluble inclusion bodies(or refractile bodies) depending on such factors as redox environment of the host cell, the expression level and the nature of the protein. There also exists a borderline case wherein both soluble and insoluble forms of proteins are expressed simultaneously. The proteins expressed in the form of inclusion bodies must be solubilized and renaturized to obtain biologically active proteins and there have been developed several methods for the renaturization of proteins produced in the form of inclusion bodies.
A common method utilizes a high concentration of denaturing chaotropic agents, e.g., urea and guanidine salts, in solubilizing inclusion bodies and the solubilized proteins are renatured after diluting or ultrafiltering the solution to lower the concentration of the denaturing agent. In this method, the solubilizing process is carried out at a high concentration, e.g., 5 to 9 M, of the denaturing agent and the renaturing process, at a lower concentration, e.g, 1 to 5 M(see, e.g., European Patent No. 0 114 506). Further, it has been reported that the addition of a small amount of an alcohol to the solution containing the denaturing agent enhances the solubilization and refolding rates(see, U.S. Pat. No. 5,109,117).
A second method carries out the solubilization of inclusion bodies at a high pH, wherein a protein is denatured, by adding an alkali such as NaOH to a solubilizing solution, and renaturing the protein by lowering the pH(see, e.g., Korean Patent No. 86911). This method may be effectively combined with the above mentioned common method to loosen the strong non-covalent attractions between the proteins in inclusion bodies.
A third method employs a *surfactant for the solubilization of inclusion bodies(see, e.g., U.S. Pat. No. 5,023,323). The hydrophobic portion of the surfactant dissipates the attractive force between proteins in the inclusion bodies by interacting strongly with the proteins, while the hydrophilic portion thereof gets in close contact with water. Thus, a surfactant solubilizes the inclusion bodies by attaching itself to protein monomers in an aqueous solution to form a stable structure. However, this method has the problem that it is difficult to remove the strongly interacting surfactant from renatured proteins.
Protein inclusion bodies which have isoelectric points generally at around neutral pH are formed when protein monomers having no net charge aggregate at a neutral pH due to strong non-covalent attractions therebetween. A denaturing agent when present at a high concentration offsets the non-covalent attractions, thereby solubilizing the inclusion bodies. Further, at a highly alkaline pH, amino acid residues of proteins become charged, thereby further facilitating solubilization of the inclusion bodies.
When a protein having cysteine residues is placed at a sufficiently high pH for the dissociation of the free SH groups of the cysteine residues, oxidation by a mild oxidant such as air occurs slowly to form intramolecular disulfide bonds between the cysteine groups. However, undesirable intermolecular disulfide bonds may also form between two or more protein monomers resulting in the formation of protein dimers, trimers and other polymeric forms.
In fact, the known methods for the renaturation of somatotropin are hampered by the problem that the proportion of the dimeric and polymeric forms of somatotropin is relatively high and, accordingly, there exists a need to develop an effective method to produce biologically active somatotropin at a high yield while minimizing the formation of the dimeric and polymeric forms of somatotropin. As it is difficult to remove the inactive dimeric and polymeric forms of somatotropin in a purification process, it is important to suppress the formation thereof in the refolding and reoxidation process. The present inventors have unexpectedly discovered that the use of a concentrated alcohol solution containing no chaotropic agents, particularly an aqueous isopropyl alcohol or n-propyl alcohol solution in the solubilizing process significantly reduces the formation of the dimeric and polymeric forms of somatotropin.